The present invention is directed to a viral vector that can introduce a desired nucleic acid sequence into a targeted host cell by retroviral infection where the nucleic acid sequence replicates episomally. Preferably, the viral vector is a lentiviral vector that also contains a heterlogous viral origin of replication (ori) and a second gene that functions as a replication transactivator.
In recent years considerable effort has been directed at applying gene delivery techniques. That term describes a wide variety of methods using recombinant biotechnology techniques to deliver a variety of different materials to a cell. These methods include, for example, vectors such as viral vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene gun, catheters, etc. The different techniques used depend in part upon the gene being transferred and the purpose therefore. Thus, for example, there are situations where only a short-term expression of the gene is desired in contrast to situations where a longer term, even permanent expression of the gene is desired.
Vectors that have been looked at include both DNA viral vectors and RNA viral vectors. For example, DNA vectors include pox vectors such as orthopox or avipox vectors (see, e.g., U.S. Pat. No. 5,656,465), herpes virus vectors, such as herpes simplex I Virus (HSV) vector [Geller, A. I. et al., J. Neurochem. 64:487 (1995); Lim, F., et al., DNA Cloning: Mammalian Systems, D. Glover, Ed., Oxford Univ. Press, Oxford, England (1995); Geller, A. I. et al., Proc. Natl. Acad. Sci., U.S.A. 90:7603 (1993); Adenovirus vectors [Legal Lasalle et al., Sci. 259-988 (1993); Davidson et al., Nat. Genet. 3:219 (1993); Yang et al., J. Virol., 69:2004 (1995); and Adeno Associated Virus Vectors [Kaplitt, M. G., et al.]; Nat. Genet. 8;148 (1994)]. Retroviral vectors include moloney murine leukemia viruses (MMLV) and human immunodeficiency viruses (HIV) [See, U.S. Pat. No. 5,665,577].
For example, a retroviral vector can be used to infect a host cell and have the genetic material integrated into that host cell with high efficiency. One example of such a vector is a modified moloney murine leukemia virus (MMLV), which has had its packaging sequences deleted to prevent packaging of the entire retroviral genome. However, that retrovirus does not transduce resting cells. Additionally, since many retroviruses typically enter cells via receptors, if the specific receptors are not present on a cell or are not present in large enough numbers, the infection is either not possible or is inefficient. Concerns have also been expressed as a result of outbreaks of wild-type viruses from the recombinant MMLV producing cell lines, i.e., reversions.
Recently, attention has focused on lentiviral vectors such as those based upon the primate lentiviruses, e.g., human immunodeficiency viruses (HIV) and simian immunodeficiency virus (SIV). HIV vectors can infect quiescent cells in addition to dividing cells. Moreover, by using a pseudotyped vector (i.e., one where an envelope protein from a different species is used), problems encountered with infecting a wide range of cell types can be overcome by selecting a particular envelope protein based upon the cell you want to infect. Moreover, in view of the complex gene splicing patterns seen in a lentiviruses such as HIV, multivalent vectors (i.e., those expressing multiple genes) having a lentiviral core, such as an HIV core, are expected to be more efficient. These vectors like MMLV also result in having the genetic material integrated into the host cell with high efficiency.
Variations in the lentiviral vectors can be made where multiple modifications are made, such as deleting nef, rev, vif and vpr genes. One can also have the 3xe2x80x2 and 5xe2x80x2 U3 deleted LTRs.
While those vectors provide many advantages, one of their prime advantagesxe2x80x94the ability to stably integrate into a host cell""s chromosomesxe2x80x94can also be a major safety concern. This ability to integrate into a chromosome can cause insertional mutagenesis [Shiramiza, B., et al., Cancer Res., 54:2069-2072 (1994); Verma, I. M. and Somia, N., Nature, 389:239-242 (1997)]. One method of dealing with this problem has been to fuse a specific DNA binding domain to the integrase (IN) polypeptide to direct integration into specific DNA sequences [Bushman, F., Science, 267:1443-1444 (1995); Bushman, F. and Miller, M. D., J. Virol., 71:458-464 (1997); Katz, R. A., et al., Virology, 217:178-190 (1996)]. However, additional improvements are still useful.
Further, there are many instances where one does not want to have a gene stably integrated, but only expressed for a limited time period. For example, such as approach is useful with xe2x80x9csuicide therapyxe2x80x9d where the gene product is designed to negatively impact the integrity of the host cell. It is also useful with angiogenesis proteins. These proteins can promote wound healing, growth of blood vessels, etc. Thus, they can be useful in dealing with individuals having circulatory problems, heart problems etc. However, these proteins can also cause the growth of blood vessel regulated tumors. Accordingly, while some expression of the protein can be beneficial, its unlimited expression can ultimately cause more harm than benefit.
One type of expression where a gene is not integrated into a chromosome is episomal replication. Adenovirus, which replicates episomally, is the most widely used nonintegrating viral vector. It produces very high titers and has a broad target range. However, that broad target range is a disadvantage in using xe2x80x9csuicide therapyxe2x80x9d. It would be desirable to have a method of gene therapy where one can target specific cells. In addition, adenoviruses, have immunogenicity problems [Haddada, et al., Curr. Top. Microbiol. Immunol., 199:297-306 (1995); Verma and Somia, Nature, 389, supra.]. Thus, instances where repeated use of the vector is necessary are problematic. SV40-based vectors also replicate episomally, and have been looked at for use in suicide therapy [Cooper, M. J., et al., Proc. Natl. Acad. Sci. USA, 94:6450-6455 (1997)]. However, these vectors are currently introduced into cells by transfection, typically ex vivo. It would be desirable to have an alternative episomal replicating vector, that can readily be introduced into a cell.
We have now discovered a viral vector system that takes advantage of retroviral infection to bring a desired nucleic acid sequence to a targeted host cell without resulting in stable integration.
In one preferred embodiment the plurality of vectors used, include lentiviral vectors. These lentiviral vectors preferably contain a selectable marker.
The lentivirus vectors include, for example, primate lentiviruses such as human immunodeficiency virus (HIV) (e.g. HIV-1 and HIV-2) and simian immunodeficiency virus (SIV); feline immunodeficiency virus (FIV); or visna virus. The primate lentiviruses show a remarkable ability to utilize a range of heterologous envelopes resulting in pseudotyped virions.
The lentiviral virion (particle) is expressed by a vector system encoding the necessary viral proteins to produce a virion (viral particle). Preferably, there is at least one vector containing a nucleic acid sequence encoding the lentiviral pol proteins necessary for reverse transcription, operably linked to a promoter. Although the vector encoding pol sequences should not encode an IN capable of integration, it preferably does encode an IN.
For example, this can be accomplished by the substitution of sites that bind to DNA, negating the peptides ability to bind to DNA and integrate. For instance, substituting Gln for the third IN wild type active site, such as in HIV-1 (E152Q). This can be done by PCR introducting the Q into N/N yielding N/N/Q. Other changes can also be carried out. For example, if a triple leucine mutant is made it will require 9 nucleotide changes to revert all three amino acids to wild type (WT) IN. Particularly preferred are mutations to the IN-DNA binding site. These are frequently known, or can readily be determined by site-directed mutageneis. In HIV-1 such active sites include 152, 64, 116, 62, 148 and 155 [Engleman, A., et al., J. Virol., 71:3507-3514 (1997); Gerton, J. L., et al., J. Virol., 72:5046-5055 (1998)]. Preferably, one makes the most changes in these sites without substantially reducing viral replication. This can readily be determined by using known in vitro assays.
There is also a vector containing a nucleic acid sequence encoding the lentiviral gag proteins necessary for forming a viral capsid operably linked to a promoter. Preferably, this gag nucleic acid sequence is on a separate vector than at least some of the pol nucleic acid sequence, still more preferably it is on a separate vector from all the pol nucleic acid sequences that encode pol proteins.
In one embodiment, the gag sequence does not express a functional MA protein, i.e. the vector can still transduce cells in the absence of the entire MA or a portion thereof, if a myristylation anchor is provided. This can be accomplished by inactivating the xe2x80x9cgenexe2x80x9d encoding the MA by additions, substitutions or deletions of the MA coding region. Preferably, this is done by deletion. Preferably, at least 25% of the MA coding region is deleted, more preferably, at least 50% is deleted, still more preferably, at least 60%, even more preferably at least 75%, still more preferably, at least 90%, yet more preferably at least 95% and most preferably the entire coding region is deleted. However, in that embodiment, a myristylation anchor (sequence) is still required. Preferably, the myristylation sequence is a heterologous (i.e., non-lentiviral) sequence.
In another embodiment the lentiviral vector is another form of self-inactivating (SIN) vector as a result of a deletion in the 3xe2x80x2 long terminal repeat region (LTR). Preferably, the vector contains a deletion within the viral promoter. The LTR of lentiviruses such as the HIV LTR contains a viral promoter. Although this promoter is relatively inefficient, when transactivated by e.g. tat, the promoter is relatively efficient. However, the presence of the viral promoter can interfere with heterologous promoters operably linked to a transgene. To minimize such interference and better regulate the expression of transgenes, the lentiviral promoter is preferably deleted.
Preferably, the vector contains a deletion within the viral promoter. The viral promoter is in the U3 region of the 3xe2x80x2 LTR. A preferred deletion is one that is 120 base pairs between Sca I and Pvu I sites, e.g. corresponding to from nucleotides 9398-9518 of HIV, encompassing the essential core elements of the HIV-1 LTR promoter (TATA box, SP 1 and NK-Kb binding sites). After reverse transcription, the deletion is transferred to the 5xe2x80x2 LTR, yielding a vector/provirus that is incapable of synthesizing vector transcripts from the 5xe2x80x2 LTR in the next round of replication. Thus, the vector of the present invention contains no mechanism by which the virus can replicate as it cannot express the viral proteins.
In another embodiment the vector is a tat deleted vector. This can be accomplished by inactivating at least the first exon of tat by known techniques such as deleting it. Alternatively, one can extend the U3 LTR deletion into the R region to remove the TAR element.
Variations can be made where the lentiviral vector has multiple modifications as compared to a wildtype lentivirus. For example, with HIV being nefxe2x88x92, revxe2x88x92, vifxe2x88x92 and vprxe2x88x92. In addition one can have MAxe2x88x92 gag, 3xe2x80x2 and 5xe2x80x2 U3 deleted LTR and variations thereof as well as the IN-inactivation.
The above-mentioned vector(s) do not contain nucleotides from the lentiviral genome that package lentiviral RNA, referred to as the lentiviral packaging sequence. In HIV this region corresponds to the region between the 5xe2x80x2 major splice donor and the gag gene initiation codon (nucleotides 301-319).
The gag and pol vector(s) forming the particle preferably do not contain a nucleic acid sequence from the lentiviral genome that expresses an envelope protein. Preferably, a separate vector contains a nucleic acid sequence encoding an envelope protein operably linked to a promoter. This env vector also does not contain a lentiviral packaging sequence. In one embodiment the env nucleic acid sequence encodes a lentiviral envelope protein.
In another embodiment the envelope protein is not from the same lentivirus as the gag and pol, but from a different virus. The resultant particle is referred to as a pseudotyped particle. By appropriate selection of envelopes one can target and xe2x80x9cinfectxe2x80x9d virtually any cell. For example, one can use an env gene that encodes an envelope protein that targets an endocytic compartment such as that of the influenza virus, VSV-G, alpha viruses (Semliki forest virus, Sindbis virus), arenaviruses (lymphocytic choriomeningitis virus), flaviviruses (tick-borne encephalitis virus, Dengue virus), rhabdoviruses (vesicular stomatitis virus, rabies virus), and orthomyxoviruses (influenza virus). Other envelope proteins that can preferably be used include those from Moloney Leukemia Virus such as MLV-A, MLV-E and GALV. These envelopes are preferably when the host cell is a primary cell.
The preferred lentivirus is a primate lentivirus [U.S. Pat. No. 5,665,577] or a feline immunodeficiency virus (FIV) [Poeschla, E. M., et al., Nat. Medicine 4:354-357 (1998)] The pol/gag nucleic acid segment(s) and the env nucleic acid segment will when expressed produce an empty lentiviral particle. By making the above-described modifications such as inactivating IN, deleting, the MA coding region, or the U3 region of the LTR, the possibility of a reversion to a wild type virus has been reduced.
A desired family of heterologous nucleic acid segment (sometimes referred to as the target molecule) can be inserted into the empty lentiviral particles by use of a plurality of vectors each containing a nucleic acid segment of interest and a lentiviral packaging sequence necessary to package lentiviral RNA into the lentiviral particles (the packaging vector). Preferably, the packaging vector contains a 5xe2x80x2 and 3xe2x80x2 lentiviral LTR with the desired nucleic acid segment inserted between them. The nucleic acid segment can be antisense molecules or more preferably, encodes a protein such as an antibody. The packaging vector preferably contains a selectable marker. These are well known in the art and include genes that change the sensitivity of a cell to a stimuli such as a nutrient, an antibiotic, etc. Genes include those for neo, puro, tk, multiple drug resistance (MDR), etc. Other genes express proteins that can readily be screened for such as green fluorescent protein (GFP), blue fluorescent protein (BFP), luciferase, LacZ, nerve growth factor receptor (NGFR), etc.
The packaging vector also contains at least one component of the episomal replicon. Preferably, one uses a DNA viral replicon. DNA viruses that can be used include SV40 Epstein-Barr virus (EBV) and BK virus [Cooper, M. J., et al., Proc. Natl. Acad. Sci. USA, 94, supra; Eckhart, W., Virology 38:120-125 (1969); Asconzioni, F., et al., Cancer Lett., 118:135-142 and Fried, M., Proc. Natl. Acad. Sci. USA, 53:486-491 (1965)]. The replicon comprises a viral DNA origin of replication (ori) and a protein that acts as a replication transactivator. Typically, that protein is an early gene product from the same virus. However, other constructs can be used. [See for example, Piechaczek, C., et al., Nucleic Acids Research, 27:426-428 (1999)]. Because the viral proteins such as large T-antigen for SV40, EBNA-1 for EBV, and large T-antigen for BK virus are transforming (i.e. tumorigenic), the use of modified constructs is preferred. For example, deleting domains that bind human tumor suppressor gene products such as p53, retinoblastoma and p107. One such construct is the SV40 mutant 107/402-T which encodes a lysine instead of glutamic acid at codon 107 and glutamic acid instead of aspartic acid at codon 402. Other amino acids can also be substituted. Binding activity can readily be determined in an in vitro assay by known means. Another construct that can be used instead of SV40 T-antigen is the S/MAR (scaffold/matrix attached region) fragment from a gene such as the human interferon xcex2-gene.
These regions are typically about 70%, A/T-rich sequences and are often associated with chromosomal origins of bidirectional replication [Piechaczek, C., Nucleic Acids Research, supra; Bode, J., Science, 255:195-197 (992); Luderus, M. E., et al., Mol. Cell Biol., 14:6297-6305 (1994)].
Both the ORI and the transactivating protein can be on the packaging vector. Alternatively, the transactivating protein can be on a separate vector.
In one embodiment, the vector containing the gene encoding the transactivating protein is not added to the host cell at the same time, but only at a later timexe2x80x94in a manner analogous to use of an inducible promoter.
The advantage of such a method is to minimize the phenomenon of xe2x80x9cgene silencingxe2x80x9d that is sometimes encountered. Other methods to avoid this problem can also be used.
For example, when an inducible promoter is used with the target molecule, minimal selection pressure is exerted on the transformed cells for those cells where the target molecule is xe2x80x9csilencedxe2x80x9d. If one also uses a marker gene, the identification of cells displaying the marker also identifies cells that can express the target molecule. If an inducible promoter is not used, it is sometimes preferable to use a xe2x80x9cforced-expressionxe2x80x9d system where the target molecule is linked to the selectable marker by use of an internal ribosome entry site (IRES) (see Marasco et al., PCT/US96/16531).
IRES sequences are known in the art and include those from encephalomycarditis virus (EMCV) [Ghattas, I. R. et al., Mol. Cell. Biol., 11:5848-5849 (1991); BiP protein [Macejak and Sarnow, Nature, 353:91 (1991)]; the Antennapedia gene of drosophilia (exons d and e) [Oh et al., Genes and Development, 6:1643-1653 (1992)]; those in polio virus [Pelletier and Sonenberg, Nature, 334:320325 (1988); see also Mountford and Smith, TIG, 11:179-184 (1985)].
Inducible promoters include those using the lac repressor from E. coli as a transcription modulator to regulate transcription from lac operator-bearing mammalian cell promoters [Brown, M. et al., Cell, 49:603-612 (1987)], those using the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. See Miller, N. and Whelan, J., Human Gene Therapy, 8:803-815 (1997). Other systems include FK506 dimer, VP16 or p65 using astradiol, RU486, diphenol murislerone or rapamycin [see Miller and Whelan, supra at FIG. 2]. Inducible systems are available from Invitrogen, Clontech and Ariad. Systems using a repessor with the operon are preferred. Regulation of transgene expression in target cells represents a critical aspect of gene therapy. For example, the lac repressor from Escherichia coli can function as a transcriptional modulator to regulate transcription from lac operator-bearing mammalian cell promoters [M. Brown et al., Cell, 49:603-612 (1987)]; Gossen and Bujard (1992); [M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)] combined the tetracycline repressor (tetr) with the transcription activator (VP16) to create a tetR-mammalian cell transcription activator fusion protein, tTa (tetR-VP 16), with the tetO-bearing minimal promoter derived from the human cytomegalovirus (hCMV) major immediate-early promoter to create a tetR-tet operator system to control gene expression in mammalian cells. Recently Yao and colleagues [F. Yao et al., Human Gene Therapy, supra] demonstrated that the tetracycline repressor (tetR) alone, rather than the tetR-mammalian cell transcription factor fusion derivatives can function as potent trans-modulator to regulate gene expression in mammalian cells when the tetracycline operator is properly positioned downstream for the TATA element of the CMVIE promoter. One particular advantage of this tetracycline inducible switch is that it does not require the use of a tetracycline repressor-mammalian cells transactivator or repressor fusion protein, which in some instances can be toxic to cells [M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); P. Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)], to achieve its regulatable effects. Preferably, the repressor is linked to the target molecule by an IRES sequence. Preferably, the inducible system is a tetR system. More preferably the system has the tetracycline operation downstream of a promoter""s TATA element such as with the CMVIE promoter.